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* Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan;
Department of Microbiology and Immunology, Nihon University School of Density at Matsudo, Matsudo, Japan; and
Department of Microbiology and Immunology, Division of Mucosal Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-deficient intercrossed Rag2 double knockout mice (LT–/– x Rag2–/–), which lack mesenteric lymph nodes and Peyer’s patches, are transferred with CD4+CD45RBhigh T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LT+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells. Of note, when a mixture of splenic CD4+CD25+ TREG cells and CD4+CD45RBhigh T cells are transferred into LT–/– x Rag2–/– recipients, CD4+CD25+ TREG cells migrate into the colon and prevent the development of colitis in LT–/– x Rag2–/– recipients as well as in the control LT+/+ x Rag2–/– recipients. These results suggest that the intestinal LP harboring CD4+CD25+ TREG cells contributes to the intestinal immune suppression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The gut-associated lymphoid tissue can be divided into effector sites, which consist of lymphocytes scattered throughout the epithelium and lamina propria (LP) of the mucosa and organized lymphoid tissues (inductive sites) that are responsible for the induction phase of the immune response (1, 9). These include Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and isolated lymphoid follicles (ILFs). It is thought that presentation of Ags to immune naive and effector cells is concentrated at these inductive sites of organized mucosal lymphoid follicles, and thus APCs tune the delicate balance between intestinal immune tolerance and inflammation.
In addition to the inductive sites for the development of colitis, however, it also remains unclear where CD4+CD25+ TREG cells suppress the development of colitis. Although it is reasonable to hypothesize that mechanisms for the induction, maintenance, and suppression of colitis would be centrally controlled by CD4+CD25+ TREG cells in the inductive sites, we question in this study whether these inductive sites are solely involved in the induction and suppression of intestinal inflammation because we recently demonstrated that human intestinal LP CD4+CD25bright T cells as well as peripheral CD4+CD25bright T cells obtained from normal individuals possess TREG activity in vitro (10). Consistent with our previous report, it has been recently reported that CD4+CD25+ TREG cells were detected in peripheral tissues and at sites of ongoing immune responses such as synovial fluid from rheumatoid arthritis patients (11), tumors (12), transplants (13), skin lesions in mice infected with Leishmania major (14), lungs from mice infected with Pneumocystis carinii (15), islets of Langerhans in diabetes models (16), and lesions in delayed-type hypersensitivity models (17) as well as in inflamed mucosa in colitic mice (8, 18). In the present study, we conducted a series of the adoptive transfer experiments focusing on intestinal LP CD4+CD25+ T cells to understand where and how CD4+CD25+ TREG cells control the mucosal immune system in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Female BALB/c, C.B-17 SCID, and C57BL/6-Ly5.2 mice were purchased from Japan CLEA. C57BL/6-Ly5.1 and C57BL/6-Ly5.2 Rag2-deficient (Rag2–/–) mice were obtained from Taconic Farms. Ly5.2 background lymphotoxin (LT)-deficient (LT–/–) mice were purchased from The Jackson Laboratory. LT–/– mice were intercrossed into Rag2–/– mice to generate LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice in the Animal Care Facility of Tokyo Medical and Dental University. Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Tokyo Medical and Dental University. Donors and littermate recipients were used at 6–12 wk of age. All experiments were approved by the regional animal study committees and were done according to institutional guidelines and Home Office regulations.
Abs and reagents
The following mAbs except DTA-1, biotinylated anti-mouse glucocorticoid-induced TNFR (GITR; eBioscience) and FJK-16s, PE-conjugated anti-mouse Foxp3 (eBioscience) were obtained from BD Pharmingen for purification of cell populations and flow cytometry analysis: PE-conjugated anti-mouse CD4 (RM4-5), PE-Cy5- and allophycocyanin-conjugated anti-mouse CD4 (L3T4), FITC-conjugated anti-mouse CD25 (7D4), PE-conjugated anti-mouse CD25 (PC61), PE-conjugated anti-mouse CD103 (E integrin) (M290), PE-conjugated anti-mouse 4
7 integrin (DATK32), PE-conjugated anti-CTLA-4 (UC10-4F10-11), FITC-conjugated anti-mouse CD45RB (16A), FITC-conjugated anti-mouse Ly5.1 (CD45.1, A20), FITC-conjugated anti-mouse Ly5.2 (CD45.2, 104), and FITC- and PerCP-conjugated anti-mouse CD3 (145-2C11). Biotinylated Abs were detected with PE- or CyChrome-streptavidin (BD Pharmingen).
Purification of T cell subsets
CD4+ T cells were isolated from normal spleen and colon using the anti-CD4 (L3T4) MACS system (Miltenyi Biotec) according to the manufacturer’s instructions. To isolate normal LP CD4+ T cells, the entire length of colon was opened longitudinally, washed with PBS, and cut into small pieces. The dissected mucosa was incubated with Ca2+, Mg2+-free HBSS containing 1 mM DTT (Sigma-Aldrich) for 45 min to remove mucus and then treated with 2.0 mg/ml collagenase and 0.01% DNase (both Worthington Biomedical) for 2 h. The cells were pelleted two times through a 40% isotonic Percoll solution, and then subjected to Ficoll-Hypaque density gradient centrifugation (40%/75%). Enriched CD4+ T cells from the spleen and the colon (spleen, 94–97% pure; colon, 80–90%, as estimated by FACSCalibur (BD Biosciences)) were then labeled with PE-conjugated anti-mouse CD4 (RM4-5), FITC-conjugated anti-CD45RB (16A), and FITC-conjugated anti-CD25 (7D4). Subpopulations of CD4+ cells were generated by two-color sorting on FACSVantage (BD Biosciences). All populations were >98.0% pure on reanalysis.
In vivo experimental design
A series of in vivo experiments was conducted to investigate the role of intestinal LP CD4+CD25+ T cells in the suppression of murine chronic colitis. In Experiment 1, to assess the role of intestinal LP CD4+CD25+ T cells obtained from normal mice in the protection of colitis, we transferred 3 x 105 splenic CD4+CD45RBhigh T cells from normal BALB/c mice with or without 1 x 105 intestinal LP CD4+CD25+ T cells into syngeneic C.B-17 SCID mice. All recipient SCID mice were sacrificed at 7 wk after transfer. In Experiment 2, to assess the necessity of gut-associated lymphoid tissues including MLNs in the development and the protection of colitis, we transferred 3 x 105 splenic CD4+CD45RBhigh T cells from normal C57BL/6-Ly5.2 mice with or without 1 x 105 splenic CD4+CD25+ TREG cells from C57BL/6-Ly5.1 mice into LT–/– x Rag2–/– mice and the control LT+/+ x Rag2–/– mice. In Experiment 3, to exclude a possible role of spleen in the suppression of colitis in addition to MLNs, we transferred 3 x 105 colitogenic LP CD4+ T cells (Ly5.2+) obtained from established CD4+CD45RBhigh T cell-transferred mice (19) with or without 3 x 105 splenic CD4+CD25+ TREG cells from C57BL/6-Ly5.1 mice into splenectomized LT–/– x Rag2–/– and LT+/+ x Rag2–/– mice.
Disease monitoring and clinical scoring
The recipient mice, after T cell transfer, were weighed initially and then three times per week thereafter. They were observed for clinical signs of illness: hunched over appearance, piloerection of the coat, diarrhea, and blood in the stool. Mice were sacrificed at the indicated time point and assessed for a clinical score that is the sum (0–8 points) of four parameters as follows: 0 or 1, hunching and wasting; 0–3, colon thickening (0, no colon thickening; 1, mild thickening; 2, moderate thickening; 3, extensive thickening); 0–3, stool consistency (0, normal beaded stool; 1, soft stool; 2, diarrhea; 3, bloody stool); and an additional point was added if gross blood was noted (19). To monitor the clinical sign during the observed period over time, the disease activity index is defined as the sum (0–5 points) of the described parameters except colon thickening.
Histological examination and immunohistology
Tissue samples were fixed in PBS containing 6% neutral-buffered formalin. Paraffin-embedded sections (5 µm) were stained with H&E. The sections were analyzed without prior knowledge of the type of T cell reconstitution and recipients. The area most affected was graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of a previously described scoring system (19). To detect CD11c+ dendritic cells and CD4+ T cells in the LP, consecutive cryostat sections (6 µm) were fixed and stained with the following rat Abs: purified CD4 (L3T4) and biotinylated anti-CD11c (HL3) (BD Pharmingen). Alexa Fluor 594 goat anti-rat IgG and streptavidin-Alexa Fluor 488 (Molecular Probes) were used as second Abs. All confocal microscopy was conducted on a BioZERO BZ8000 (Keyence).
Flow cytometry
To detect the surface expression of a variety of molecules, isolated splenocytes or LP mononuclear cells were preincubated with an Fc
R-blocking mAb (CD16/32, 2.4G2; BD Pharmingen) for 20 min followed by incubation with specific FITC-, PE-, PE-Cy5-, or biotin-labeled Abs for 30 min on ice. Biotinylated Abs were detected with PE- or CyChrome-streptavidin. Intracellular Foxp3 staining was performed with the PE anti-mouse Foxp3 staining set (eBioscience) according to the manufacturer’s instructions. Standard two- or three-color flow cytometric analyses were obtained using the FACSCalibur method and CellQuest software. Background fluorescence was assessed by staining with control irrelevant isotype-matched mAbs.
Cytokine ELISA
To measure cytokine production, 1 x 105 LP CD4+ T cells were cultured in 200 µl of culture medium at 37°C in a humidified atmosphere containing 5% CO2 in 96-well plates (Costar) precoated with 5 µg/ml hamster anti-mouse CD3
mAb (145-2C11; BD Pharmingen) and 2 µg/ml hamster anti-mouse CD28 mAb (37.51; BD Pharmingen) in PBS overnight at 4°C. Culture supernatants were removed after 48 h and assayed for cytokine production. Cytokine concentrations were determined by specific ELISA per the manufacturer’s recommendation (R&D Systems).
In vitro TREG cell activity
LP mononuclear cells and splenocytes from normal BALB/c mice were separated into unfractioned CD4+ T cells, CD4+CD25+ and CD4+CD25– T cells using the anti-CD4 (L3T4) MACS magnetic separation system and/or FACSVantage as described. Cells (5 x 104) and mitomycin C-treated BALB/c CD4– cells (2 x 105) as APCs were cultured for 72 h in round-bottom 96-well plates in RPMI 1640 supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME. Cells were stimulated with 1 µg/ml anti-mouse CD3 mAb. In coculture experiments, the same number of splenic CD4+CD25+ or CD4+CD25– cells, or LP CD4+CD25+ or CD4+CD25– cells (5 x 104), were added into wells with the fixed dose of splenic CD4+CD25+ responder cells (5 x 104) and mitomycin C-treated CD4– cells (2 x 105), as APCs. Incorporation of [3H]thymidine (1 µCi/well) by proliferating cells was measured during the last 9 h of culture.
Statistical analysis
The results were expressed as the mean ± SD. Groups of data were compared by Mann-Whitney U test. Differences were considered to be statistically significant for a value of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Paired samples of spleen and colon obtained from normal BALB/c mice were analyzed by flow cytometry for the presence of the CD4+CD25+ T cells. Consistent with previous reports described, in naturally occurring CD4+CD25+ TREG cells (4, 5, 6), 7.0 ± 0.5% of the splenic CD4+ T cells were CD25+ (Fig. 1A). Similarly, 7.2 ± 1.0% of the colonic LP CD4+ T cells were also CD25+ (Fig. 1A). Because we previously demonstrated that human intestinal LP CD4+CD25bright T cells obtained from healthy individuals function as TREG cells in vitro (10), we postulated that intestinal LP as well as MLNs is another important site of regulation of immune responses for intestinal homeostasis in vivo. To prove it, we first assessed whether murine intestinal LP CD4+CD25+ T cells also express well-known TREG markers, such as CTLA-4, GITR, and Foxp3. Like the control splenic CD4+CD25+ T cells, the expression of CTLA-4, GITR, and Foxp3 was markedly up-regulated in or on the intestinal LP CD4+CD25+ T cells (Fig. 1B) compared with the paired CD4+CD25– T cells. Unexpectedly, but consistent with our human study (10), colonic LP CD4+CD25– T cells expressed CTLA-4, albeit to lesser extent compared with the paired colonic CD4+CD25+ T cells (Fig. 1B). To further investigate the migration property of these CD4+CD25+ T cells, we assessed the expression of 47/E7 integrins, which are gut-homing receptors essential to migrate into the colon. As shown in Fig. 1B, 
10–30% of cells in each subpopulation expressed 47 integrin. In contrast, E7 integrin was predominantly expressed on the splenic and LP CD4+CD25+ T cells, but not on the paired CD4+CD25– T cells, indicating that a part of splenic and LP CD4+CD25+ T cells can directly migrate into the gut.
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Murine intestinal LP CD4+CD25+ T cells suppress the development of the CD4+CD45RBhigh T cell-transferred colitis
To next analyze the functional role of murine intestinal LP CD4+CD25+ T cell subset in vivo, we tested the TREG activity of the intestinal LP CD4+CD25+ T cells using the classical SCID-transferred colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells (19). C.B-17 SCID mice were injected i.p. with one or two subpopulations of sorted CD4+ T cell in PBS: 1) splenic CD4+CD45RBhigh T cells alone (3 x 105 per mouse) as a positive control, 2) splenic CD4+CD45RBhigh (3 x 105 per mouse) with splenic CD4+CD25+ T cells (1 x 105) as a negative control, and 3) splenic CD4+CD45RBhigh (3 x 105) with LP CD4+CD25+ T cells (1 x 105). The results clearly demonstrated that control of intestinal inflammation resided predominantly within the intestinal LP CD4+CD25+ subpopulation as well as the splenic CD4+CD25+ T cells, as these cells significantly inhibited the development of wasting disease (Fig. 2A) and colitis (Fig. 2, B–E). Colons from mice reconstituted with a mixture of CD4+CD45RBhigh and LP CD4+CD25+ T cells exhibited no detectable pathological changes and were indistinguishable from colons from mice reconstituted with a mixture of CD4+CD45RBhigh plus splenic CD4+CD25+ T cells (Fig. 2B). In contrast, mice reconstituted with CD4+CD45RBhigh cells alone developed wasting disease and severe colitis (Fig. 2). Totally, the assessment of colitis by clinical scores showed a clear difference among three groups (Fig. 2C). Clinical score for mice transferred with a mixture of CD4+CD45RBhigh and LP CD4+CD25+ T cells was significantly decreased as compared with that for mice transferred with CD4+CD45RBhigh T cells alone. Histological examination showed prominent epithelial hyperplasia with glandular elongation with a massive infiltration of mononuclear cells in the LP of the colon from the control mice transferred with CD4+CD45RBhigh T cells alone (Fig. 2D). In contrast, the glandular elongation was mostly abrogated and only a few mononuclear cells were observed in the colonic LP from mice reconstituted with a mixture of CD4+CD45RBhigh plus splenic or LP CD4+CD25+ T cells (Fig. 2D). This difference was also confirmed by histological scores of multiple colon sections, which were 5.63 ± 0.89 in control mice transferred with CD4+CD45RBhigh T cells alone, 1.21 ± 0.97 in mice transferred with CD4+CD45RBhigh T cells plus splenic CD4+CD25+ T cells, and 2.40 ± 1.83 in mice transferred with CD4+CD45RBhigh T cells plus LP CD4+CD25+ T cells (p < 0.05, mice transferred with CD4+CD45RBhigh T cells alone vs mice transferred with CD4+CD45RBhigh T cells plus splenic or LP CD4+CD25+ T cells) (Fig. 2E).
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, IL-2, and IL-10 production by anti-CD3/CD28 mAb-stimulated LP CD4+ T cells. As shown in Fig. 3B, production of Th1 cytokines (IFN-, IL-2) was significantly reduced in LP CD4+ T cells from the mice transferred with CD4+CD45RBhigh plus LP or splenic CD4+CD25+ T cells as compared with those transferred with CD4+CD45RBhigh T cells alone (p < 0.05). In contrast, production of IL-10 was not significantly affected among the groups (Fig. 3B).
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–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells
To further investigate the origin of LP CD4+CD25+ TREG cells and their role in suppressing the development of colitis, we generated LT–/– x Rag2–/– mice, which lack conventional lymphoid tissues (inductive sites) including MLNs, PPs, and ILFs, as recipients for the adoptive transfer experiments. We excluded the impact of these inductive sites because it was possible that it is essential for LP CD4+CD25+ TREG cells to be instructed to differentiate to gut-homing LP TREG cells in these inductive sites. Before addressing this issue, we first transferred splenic CD4+CD45RBhigh T cells from normal C57BL/6 mice into LT–/– x Rag2–/– mice and the littermate control LT+/+ x Rag2–/– mice to assess the role of MLNs as inductive sites in inducing colitis. When CD4+CD45RBhigh T cells were transferred into the control LT+/+ x Rag2–/– mice, expectedly, the recipients rapidly developed severe wasting disease associated with clinical signs of severe colitis, in particular, weight loss, persistent diarrhea and occasionally also bloody stool and anal prolapses (Fig. 4A). When CD4+CD45RBhigh T cells were transferred into the LT–/– x Rag2–/– mice, however, the recipients also developed severe wasting chronic colitis despite the delayed onset and kinetics (Fig. 4A). Clinical scores in these mice eventually reached almost the same with those in LT+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells 10 wk after transfer (Fig. 4A). The rapid onset of colitis in the recipient LT+/+ x Rag2–/– mice could easily be explained by the existence of MLNs in these mice and migration of effector CD4+ cells primed in the these sites into the colon, but the evidence that the recipient LT–/– x Rag2–/– mice, albeit delayed, developed colitis indicates that there must be other sites where CD4+ T cells could be primed besides the MLNs. These LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells had an enlarged colon with a significantly thickened wall 10 wk after the transfer (data not shown). At the autopsy of mice, we confirmed that our established LT–/– x Rag2–/– mice macroscopically lacked MLNs (Fig. 4B) and other peripheral LNs (data not shown) in contrast to control LT+/+ x Rag2–/– mice (Fig. 4B). Tissue sections from LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells were characterized by inflammatory infiltrate, epithelial hyperplasia, crypt cell damage, and goblet cell depletion (Fig. 4C).
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+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells and splenic CD4+CD25+ TREG cells did not show weight loss and clinical signs of colitis throughout the entire observation period (Fig. 4A). Of note, LT–/– x Rag2–/– mice transferred with a mixture of CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells also did not manifest clinical signs of colitis (Fig. 4A). Consistent with the lack of clinical signs of colitis, LT+/+ x Rag2–/– or LT–/– x Rag2–/– recipients cotransferred with CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells displayed no histological evidence of intestinal inflammation (Fig. 4C). The difference among each group was also confirmed by histological scoring of multiple colon sections, which was 4.85 ± 1.58 in LT+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells alone, and 1.40 ± 0.96 in LT+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells plus splenic CD4+CD25+ T cells (p < 0.05), and 5.60 ± 0.40 in LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells alone, and 0.43 ± 0.23 in LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells plus splenic CD4+CD25+ T cells (p < 0.05) (Fig. 4D).
We also examined the cytokine production by LP CD4+ T cells from each group of mice. As shown in Fig. 5, LP CD4+ cells from the LT+/+ x Rag2–/– and LT–/– x Rag2–/– recipients transferred with CD4+CD45RBhigh T cells alone produced significantly higher amount of IFN- and IL-2 as compared with those transferred with CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells upon in vitro anti-CD3/CD28 mAbs stimulation. In contrast, the production of IL-10 was not significantly affected.
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+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with a mixture of CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells 10 wk after transfer (Fig. 6A) compared with those in the paired recipients transferred with CD4+CD45RBhigh T cells alone. To further assess the role of LP as inductive and/or suppressive site, the expression of CD11c in the colon was investigated by immunohistochemistry (Fig. 6B). Immunohistochemical analysis of the colons revealed that significant numbers of CD11c+ dendritic cells were surrounded by many CD4+ T cells in both in LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells alone, but with few CD4+ T cells and CD11c+ cells in LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with a mixture of CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells (Fig. 6B), suggesting a possible role of LP as a site for actively interacting between CD11c+ dendritic cells and CD4+ T cells.
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–/– x Rag2–/– mice
To next assess the in vivo expansion of CD4+CD45RBhigh T cells and CD4+CD25+ TREG cells after adoptive transfer at a 3:1 ratio (CD4+CD45RBhigh (Ly5. 2+) to CD4+CD25+ T cells (Ly5.1+)), colonic LP CD4+ cells were analyzed for the ratio of Ly5.2- to Ly5.1-derived cells. As shown in Fig. 7A, 10–15% of total LP CD4+ T cells both in LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells plus splenic CD4+CD25+ T cells were derived from Ly5.1+CD4+CD25+ T cells in the colon. These results suggested that splenic CD4+CD25+ T cells migrated to the colon, and prevented the development of colitis primarily by inhibiting the expansion and/or infiltration of pathogenic CD4+ T cells in the colon and secondarily by inhibiting the development of pathogenic Th1 cells producing IFN- and IL-2.
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3–16% of splenic and LP Ly.5.2+ cells were converted into inducible CD4+Foxp3+ TREG cells in both LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells alone or with a mixture of CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells, but most Ly.5.1-derived CD4+CD25+ TREG cells (78–88%) retained Foxp3 in both LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with a mixture of CD4+CD45RBhigh T cells and splenic CD4+CD25+ T cells (Fig. 7B). Splenic CD4+CD25+ T cells suppressed the expansion of colitogenic LP CD4+ T cells in the gut
With respect to the site for suppression of effector and memory CD4+ T cells, it was also possible that naturally arising CD4+CD25+ T cells suppress the activation of CD4+CD45RBhigh T cells and the expansion of the differentiated effector CD4+ T cells in the spleen rather than in the gut. To clarify that CD4+CD25+ T cells suppress the expansion of colitogenic effector and memory CD4+ T cells in the gut, we finally transferred colitogenic LP CD4+ T cells obtained from colitic mice transferred with CD4+CD45RBhigh T cells (Ly5.2+) (19) with or without splenic CD4+CD25+ T cells (Ly5.1+) into splenectomized LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice to exclude the impact of spleen. Both splenectomized LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with colitic LP CD4+ T cells (Ly5.2+) developed wasting disease (data not shown) and colitis by assessing histological findings (Fig. 8, A–C) and the recovered CD4+ cell numbers from the LP (Fig. 8D). In contrast, splenectomized LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with a mixture of colitic LP CD4+ T cells (Ly5.2+) and splenic CD4+CD25+ T cells (Ly5.1+) at a 1:1 ratio did not develop wasting disease (data not shown) and colitis (Fig. 8, A–D). Of note, we found that 30–40% of LP CD4+ T cells in splenectomized LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice cotransferred with a mixture were derived from Ly5.1+ cells (Fig. 8E). Furthermore, we confirmed that CD4+Foxp3+ T cells residing in the LP were mostly derived from Ly5.1+ population in both splenectomized LT+/+ x Rag2–/– and LT–/– x Rag2–/– mice transferred with a mixture of colitic LP CD4+ T cells (Ly5.2+) and CD4+CD25+ T cells (Ly5.1+) (Fig. 8F), indicating that LP acts as a suppressive site, and spleen is not solely essential to act as a suppressive site to inhibit the expansion of effector CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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–/– x Rag2–/– mice, which lack MLNs, ILFs, and PPs, were transferred with CD4+CD45RBhigh T cells, they did develop severe wasting disease and colitis despite the delayed onset and kinetics as compared with the control LT+/+ x Rag2–/– mice transferred with CD4+CD45RBhigh T cells. Of note, splenic CD4+CD25+ T cells can migrate into the LP, and prevent the development of CD4+CD45RBhigh T cell-transferred colitis in MLN-null LT–/– x Rag2–/– recipient mice. These results suggest that at least in part intestinal LP CD4+CD25+ T cells without the instruction by an MLN environment directly migrate into the gut and act as TREG cells, and therefore may contribute to the intestinal immune homeostasis in vivo.
We have recently demonstrated that human CD4+CD25bright T cells resided in the intestinal LP, expressed CTLA-4, GITR, and Foxp3, and possessed TREG activity in vitro (10). Although the results indicate that these cells might serve as mucosal (nonlymphoid) TREG cells to maintain intestinal homeostasis against many luminal Ags, it was impossible to determine whether they actually suppress the development of colitis in vivo using any human studies. To answer the question, it was necessary to translate into the mouse experimental system. To address this issue, we proceeded with two approaches using the different adoptive transfer experiments in this study. We first directly assessed whether the cotransfer of murine intestinal LP CD4+CD25+ T cells isolated from normal mice suppress the development of colitis induced by the adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. As shown in Fig. 2, we found the clinical score in SCID mice transferred with CD4+CD45RBhigh T cells and intestinal LP CD4+CD25+ T cells at a ratio of 3:1 was significantly decreased as compared with that in SCID mice transferred with CD4+CD45RBhigh T cells alone, indicating that the murine intestinal LP CD4+CD25+ T cells maintain intestinal homeostasis to suppress the development of colitis in vivo. Consistent with this, murine intestinal LP CD4+CD25+ T cells expressed constitutively CTLA-4, GITR, and Foxp3 and suppressed the proliferation of responder cells in vitro, such as human LP CD4+CD25bright T cells (10). Furthermore, because we also found that LP CD4+CD25+ T cells did partially express 47 and E7 integrins, it is conceivable that these gut-homing receptor-expressing LP CD4+CD25+ T cells might migrate into the colon from outside of the gut. Although it has been reported that CD4+CD25+ TREG cells reside in nonlymphoid tissues (10, 11, 12, 13, 14, 15, 16, 17, 18), our current data now provide the first experimental evidence that intestinal LP CD4+CD25+ T cells prevent the development of colitis in vivo.
Having the evidence that the murine intestinal LP CD4+CD25+ T cells suppressed the development of colitis induced by the adoptive transfer of CD4+CD45RBhigh T cells, we next asked whether MLNs are not fully essential for the suppression of colitis by splenic CD4+CD25+ T cells because it was still possible that 1) a part of the LP CD4+CD25+ T cells was needed to be instructed in MLNs to differentiate to gut-homing receptor-expressing TREG cells (17) to migrate to the gut, and also possible that 2) the transferred LP CD4+CD25+ T cells acted as TREG cells in MLNs rather than in the intestine in the first adoptive transfer experiment (Figs. 2 and 3). To address these issues, it was important to assess the CD4+CD25+ TREG cells without the impact of MLNs, which are thought to be representative inductive and suppressive sites for classical splenic CD4+CD25+ TREG cells because high expression levels of CD62 ligand enable both naive CD4+ T cells and splenic CD4+CD25+ TREG cells to efficiently enter the Ag-draining lymph nodes from the bloodstream. As the second approach to address this issue, thus, we generated LT–/– x Rag2–/– mice as recipients for the following adoptive transfer experiment. Before starting the experiment, it was unclear whether the LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells alone develop colitis, or rather it was likely to envisage that these mice did not develop colitis because MLNs are thought to be very important as inductive sites for the development of colitis. However, it was noteworthy that these mice did develop wasting disease and colitis to a similar extent of the transferred LT+/+ x Rag2–/– mice 10 wk after transfer, although it took a longer period to establish colitis as compared with the LT+/+ x Rag2–/– recipients (Fig. 4A). Although this fact is actually not a main focus in this study, it is possible that spleen and/or LP are complimentary inductive sites to develop colitis under the absence of MLNs. Consistent with this hypothesis, it has been reported that naive T cells can recruit to the inflamed intestinal mucosa, although these cells are usually excluded from uninflamed nonlymphoid tissues (20). However, the delayed kinetics of the development of colitis in the LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells indicates that MLNs are involved in the induction of colitis by their functioning as a professional inductive site. Further study will be needed to address this initial immune response for the development of colitis.
As our focus in this study, we also found that the cotransfer of splenic CD4+CD25+ T cells obtained from normal mice prevent the development of colitis in LT–/– x Rag2–/– mice transferred with CD4+CD45RBhigh T cells as well as in LT+/+ x Rag2–/– recipients, indicating that splenic CD4+CD25+ T cells can suppress the development of colitis in the absence of MLNs. Moreover, we demonstrated that Ly5.1-CD4+CD25+ T cells resided in the colon in MLN-null LT–/– x Rag2–/– mice cotransferred with Ly5.2-derived CD4+CD45RBhigh T cells and Ly5.1-derived splenic CD4+CD25+ T cells, suggesting that the LP might be a regulatory site between colitogenic effector/memory cells and TREG cells to suppress intestinal inflammation probably as a second line of suppression (17). It was also possible, however, that CD4+CD25+ TREG cells prevented the expansion of pathogenic effector CD4+ T cells and the migration to the gut in the recipient’s spleen rather in the gut. With respect to this issue, we also demonstrated that cotransfer of splenic CD4+CD25+ T cells prevented the development of colitis induced by adoptive transfer of colitogenic LP CD4+ T cells in splenectomized LT–/– x Rag2–/– recipients (Fig. 8). Because colitogenic LP CD4+ T cells that have a phenotype of effector/memory CD4+CD44highCD62L– cells (21) should have migrated to the gut and expanded in the gut, it is very likely that splenic CD4+CD25+ T cells can directly migrate to the gut and suppress the expansion of these colitogenic CD4+ T cells in the gut. With respect to the equilibrium of pathogenic CD4+ T cells and TREG cells, however, further studies will be needed because we found that effector to TREG cell ratio varied by different experimental settings (Figs. 6 and 8).
Finally, it should be discussed the protective mechanism by CD4+CD25+ TREG cells of this SCID/Rag2–/– colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells from the standpoint of the sites of active suppression. Indeed, Mottet et al. (18) previously demonstrated that not only effector CD4+ T cells but also CD4+CD25+ TREG cells accumulate in the intestinal LP in addition to the MLNs in the cured SCID mice by retransferring splenic CD4+CD25+ T cells 3–4 wk after the first transfer of CD4+CD45RBhigh T cells, it remains to be determined whether intestinal inflammation can be suppressed solely by LP or MLN CD4+CD25+ TREG cells in this setting. In contrast, Denning et al. (8) recently demonstrated that 7 integrin-deficient (7–/–) CD4+CD25+ TREG cells that preferentially migrate to MLNs, but are impaired in their ability to migrate to the intestine because of the lack of the gut-homing 47/E7 integrin molecules, are capable of preventing intestinal inflammation, suggesting TREG accumulation in the intestine is dispensable for the protection of this colitis model. In this protection protocol, indeed, it is possible that 7+/+ CD4+CD25+ TREG cells are not needed to suppress the development of colitis because 7–/– CD4+CD25+ TREG cells directly migrate to MLNs and can inhibit naive CD4+CD45RBhigh T cell activation and proliferation within Ag-draining MLNs, resulting in suppressing the development of the gut-seeking activated effector CD4+ T cells instructed to express the gut-homing receptors such as 47/E7 intergrin. However, it still remains unknown whether mucosal CD4+CD25+ TREG cells are necessary for the suppression of mucosal pathogenic effector CD4+ T cell ex vivo especially in the therapeutic protocol that can be assessed and whether LP CD4+CD25+ TREG cells as effector TREG cells can suppress the surrounding LP effector CD4+ T cells ex vivo. In our adoptive transfer experiment using splenectomized MLN-null LT–/– x Rag2–/– mice, however, we clearly demonstrated that cotransfer of splenic CD4+CD25+ TREG cells suppressed the development of colitis despite the lack of spleen and MLNs and found that these TREG cells migrated to the effector sites, in this case, the intestine, suggesting that intestinal LP CD4+CD25+ TREG cells play an important role at least in part for the suppression of intestinal inflammation in the gut.
In conclusion, our findings showed that intestinal LP functions not only as a critical effector site for inflammatory responses but also as a regulatory (suppressive) site that CD4+CD25+ TREG cells directly control the pathogenic effector CD4+ cells as a second line of suppression (effector TREG) site together with the MLNs as a first line of suppression (naive TREG) site.
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1 This study was supported in part by grants-in-aid for Scientific Research, Scientific Research on Priority Areas, Exploratory Research and Creative Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare; the Japan Medical Association; Foundation for Advancement of International Science; Terumo Life Science Foundation; Ohyama Health Foundation; Yakult Bio-Science Foundation; and Research Fund of Mitsukoshi Health and Welfare Foundation. 
2 Address correspondence and reprint requests to Dr. Takanori Kanai, Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: taka.gast{at}tmd.ac.jp
3 Abbreviations used in this paper: TREG, regulatory T; LP, lamina propria; PP, Peyer’s patch; MLN, mesenteric lymph node; ILF, isolated lymphoid follicle; GITR, glucocorticoid-induced TNFR; LT, lymphotoxin.
Received for publication May 18, 2006. Accepted for publication January 17, 2007.
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